The present invention relates generally to isolated genes which encode polypeptides involved in cellulose biosynthesis and transgenic organisms expressing same in sense or antisense orientation, or as ribozymes, co-suppression or gene-targeting molecules. More particularly, the present invention is directed to a nucleic acid molecule isolated from Arabidopsis thaliana, Oryza sativa, wheat, barley, maize, Brassica ssp., Gossypium hirsutum and Eucalyptus ssp. which encode an enzyme which is important in cellulose biosynthesis, in particular the cellulose synthase enzyme and homologues, analogues and derivatives thereof and uses of same in the production of transgenic plants expressing altered cellulose biosynthetic properties.
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Sequence identity numbers (SEQ ID Nos.) for the nucleotide and amino acid sequences referred to in the specification are defined after the bibliography.
Throughout the specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Cellulose, the world""s most abundant biopolymer, is the most characteristic component of plant cell walls in so far as it forms much of the structural framework of the cell wall. Cellulose is comprised of crystalline xcex2-1,4-glucan microfibrils. The crystalline microfibrils are extremely strong and resist enzymic and mechanical degradation, an important factor in determining the nutritional quantity, digestibility and palatability of animal and human foodstuffs. As cellulose is also the dominant structural component of industrially-important plant fibres, such as cotton, flax, hemp, jute and the timber crops such as Eucalyptus ssp. and Pinus ssp., amongst others, there is considerable economic benefit to be derived from the manipulation of cellulose content and/or quantity in plants. In particular. the production of food and fibre crops with altered cellulose content are highly desirable objectives.
The synthesis of cellulose involves the xcex2-1,4-linkage of glucose monomers, in the form of a nucleoside diphospoglucose such as UDP-glucose, to a pre-existing cellulose chain, catalysed by the enzyme cellulose synthase.
Several attempts to identify the components of the functional cellulose synthase in plants have failed, because levels of xcex2-1,4-glucan or crystalline cellulose produced in such assays have hitherto been too low to permit enzyme purification for protein sequence determination. Insufficient homology between bacterial xcex2-1,4-glucan synthase genes and plant cellulose synthase genes has also prevented the use of hybridisation as an approach to isolating the plant homologues of bacterial xcex2-1,4-glucan (cellulose) synthases.
Furthermore, it has not been possible to demonstrate that the cellulose synthase enzyme from plants is the same as, or functionally related to, other purified and characterised enzymes involved in polysaccharide biosynthesis. As a consequence, the cellulose synthase enzyme has not been isolated from plants and, until the present invention, no nucleic acid molecule has been characterised which functionally-encodes a plant cellulose synthase enzyme.
In work leading up to the present invention, the inventors have generated several novel mutant Arabidopsis thaliana plants which are defective in cellulose biosynthesis. The inventors have further isolated a cellulose synthase gene designated RSW1, which is involved in cellulose biosynthesis in Arabidopsis thaliana, and homologous sequences in Oryza sativa, wheat, barley, maize, Brassica ssp., Gossypium hirsutum and Eucalyptus ssp. The isolated nucleic acid molecules of the present invention provide the means by which cellulose content and structure may be modified in plants to produce a range of useful fibres suitable for specific industrial purposes, for example increased decay resistance of timber and altered digestibility of foodstuffs, amongst others.
Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes, or is complementary to a sequence which encodes a polypeptide of the cellulose biosynthetic pathway or a functional homologue, analogue or derivative thereof.
The nucleic acid molecule of the invention may be derived from a prokaryotic source or a eukaryotic source.
Those skilled in the art will be aware that cellulose production requires not only the presence of a catalytic subunit, but also its activation and organisation into arrays which favour the crystallization of glucan chains. This organisation is radically different between bacteria, which possess linear arrays, and higher plants, which possess hexameric clusters or xe2x80x9crosettesxe2x80x9d, of glucan chains. The correct organisation and activation of the bacterial enzyme may require many factors which are either not known, or alternatively, not known to be present in plant cells, for example specific membrane lipids to impart an active conformation on the enzyme complex or protein, or the bacterial c-di-GMP activation system. Accordingly, the use of a plant-derived sequence in eukaryotic cells such as plants provides significant advantages compared to the use of bacterially-derived sequences.
Accordingly, the present invention does not extend to known genes encoding the catalytic subunit of Agrobacterium tumefaciens or Acetobacter xylinum or Acetobacter pasteurianus cellulose synthase, or the use of such known bacterial genes and polypeptides to manipulate cellulose.
Preferably, the subject nucleic acid molecule is derived from an eukaryotic organism.
In a more preferred embodiment of the invention, the isolated nucleic acid molecule of the invention encodes a plant cellulose synthase or a catalytic subunit thereof, or a homologue, analogue or derivative thereof.
More preferably, the isolated nucleic acid molecule encodes a plant cellulose synthase polypeptide which is associated with the primary cell wall of a plant cell. In an alternative preferred embodiment, the nucleic acid molecule of the invention encodes a plant cellulose synthase or catalytic subunit thereof which is normally associated with the secondary cell wall of a plant cell.
In a more preferred embodiment, the nucleic acid molecule of the invention is a cDNA molecule, genomic clone, mRNA molecule or a synthetic oligonucleotide molecule.
In a particularly preferred embodiment, the present invention provides an isolated nucleic acid molecule which encodes or is complementary to a nucleic acid molecule which encodes the Arabidopsis thaliana, Gossypium hirsutum (cotton), Oryza sativa (rice), Eucalyptus ssp., Brassica ssp. wheat, barley or maize cellulose synthase enzyme or a catalytic subunit thereof or a polypeptide component, homologue, analogue or derivative thereof.
As exemplified herein, the present inventors have identified cellulose biosynthesis genes in maize, wheat, barley, rice, cotton, Brassica ssp. and Eucalyptus ssp., in addition to the specific Arabidopsis thaliana RSW1 gene sequence which has been shown to be particularly useful for altering cellulose and/or xcex2-1,4-glucan and/or starch levels in cells.
Hereinafter the term xe2x80x9cpolypeptide of the cellulose biosynthetic pathwayxe2x80x9d or similar term shall be taken to refer to a polypeptide or a protein or a part, homologue, analogue or derivative thereof which is involved in one or more of the biosynthetic steps leading to the production of cellulose or any related xcex2-1,4-glucan polymer in plants. In the present context, a polypeptide of the cellulose biosynthetic pathway shall also be taken to include both an active enzyme which contributes to the biosynthesis of cellulose or any related xcex2-1,4-glucan polymer in plants and to a polypeptide component of such an enzyme. As used herein, a polypeptide of the cellulose biosynthetic pathway thus includes cellulose synthase. Those skilled in the art will be aware of other cellulose biosynthetic pathway polypeptides in plants.
The term xe2x80x9crelated xcex2-1,4-glucan polymerxe2x80x9d shall be taken to include any carbohydrate molecule comprised of a primary structure of xcex2-1,4-linked glucose monomers similar to the structure of the components of the cellulose microfibril. wherein the relative arrangement or relative configuration of the glucan chains may differ from their relative configuration in microfibrils of cellulose. As used herein, a related xcex2-1,4-glucan polymer includes those xcex2-1,4-glucan polymers wherein individual xcex2-1,4-glucan microfibrils are arranged in an anti-parallel or some other relative configuration not found in a cellulose molecule of plants and those non-crystalline xcex2-1,4-glucans described as lacking the resistance to extraction and degradation that characterise cellulose microfibrils.
The term xe2x80x9ccellulose synthasexe2x80x9d shall be taken to refer to a polypeptide which is required to catalyse a xcex2-1,4-glucan linkage to a cellulose microfibril.
Reference herein to xe2x80x9cgenexe2x80x9d is to be taken in its broadest context and includes:
(i) a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5xe2x80x2- and 3xe2x80x2-untranslated sequences); or
(ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and 5xe2x80x2- and 3xe2x80x2-untranslated sequences of the gene.
The term xe2x80x9cgenexe2x80x9d is also used to describe synthetic or fusion molecules encoding all or part of a functional product.
In the present context, the term xe2x80x9ccellulose genexe2x80x9d or xe2x80x9ccellulose genetic sequencexe2x80x9d or similar term shall be taken to refer to any gene as hereinbefore defined which encodes a polypeptide of the cellulose biosynthetic pathway and includes a cellulose synthase gene.
The term xe2x80x9ccellulose synthase genexe2x80x9d shall be taken to refer to any cellulose gene which specifically encodes a polypeptide which is a component of a functional enzyme having cellulose synthase activity i.e. an enzyme which catalyses a xcex2-1,4-glucan linkage to a cellulose microfibril.
Preferred cellulose genes may be derived from a naturally-occurring cellulose gene by standard recombinant techniques. Generally, a cellulose gene may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or additions. Nucleotide insertional derivatives of the cellulose synthase gene of the present invention include 5xe2x80x2 and 3xe2x80x2 terminal fusions as well as intra-sequence insertions of single or multiple nucleotides. Insertional nucleotide sequence variants are those in which one or more nucleotides are introduced into a predetermined site in the nucleotide sequence although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more nucleotides from the sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide inserted in its place. Such a substitution may be xe2x80x9csilentxe2x80x9d in that the substitution does not change the amino acid defined by the codon. Alternatively, substituents are designed to alter one amino acid for another similar acting amino acid, or amino acid of like charge, polarity, or hydrophobicity.
As used herein, the term xe2x80x9cderived fromxe2x80x9d shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.
For the present purpose, xe2x80x9chomologuesxe2x80x9d of a nucleotide sequence shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence within said sequence, of one or more nucleotide substitutions, insertions, deletions, or rearrangements.
xe2x80x9cAnaloguesxe2x80x9d of a nucleotide sequence set forth herein shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence of any non-nucleotide constituents not normally present in said isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radionucleotides, reporter molecules such as, but not limited to DIG, alkaline phosphatase or horseradish peroxidase, amongst others.
xe2x80x9cDerivativesxe2x80x9d of a nucleotide sequence set forth herein shall be taken to refer to any isolated nucleic acid molecule which contains significant sequence similarity to said sequence or a part thereof. Generally, the nucleotide sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions. Nucleotide insertional derivatives of the nucleotide sequence of the present invention include 5xe2x80x2 and 3xe2x80x2 terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues. Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of said sequence, although random insertion is also possible with suitable screening of the resulting product being performed. Deletional variants are characterised by the removal of one or more nucleotides from the nucleotide sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide or nucleotide analogue inserted in its place.
The present invention extends to the isolated nucleic acid molecule when integrated into the genome of a cell as an addition to the endogenous cellular complement of cellulose synthase genes. The said integrated nucleic acid molecule may, or may not, contain promoter sequences to regulate expression of the subject genetic sequence.
The isolated nucleic acid molecule of the present invention may be introduced into and expressed in any cell, for example a plant cell, fungal cell, insect cell. animal cell, yeast cell or bacterial cell. Those skilled in the art will be aware of any moficiations which are required to the codon usage or promoter sequences or other regulatory sequences, in order for expression to occur in such cells.
Another aspect of the present invention is directed to a nucleic acid molecule which comprises a sequence of nucleotides corresponding or complementary to any one or more of the sequences set forth in SEQ ID Nos:1, 3, 4, 5, 7, 9, 11, or 13, or having at least about 40%, more preferably at least about 55%, still more preferably at least about 65%, yet still more preferably at least about 75-80% and even still more preferably at least about 85-95% nucleotide similarity to all, or a part thereof.
According to this aspect of the invention, said nucleic acid molecule encodes, or is complementary to a nucleotide sequence encoding, a polypeptide of the cellulose biosynthetic pathway in a plant or a homologue. analogue or derivative thereof.
Preferably, a nucleic acid molecule which is at least 40% related to any one or more of the sequences set forth in SEQ ID Nos:1, 3, 4, 5, 7, 9, 11, or 13 comprises a nucleotide sequence which encodes or is complementary to a sequence which encodes a plant cellulose synthase, more preferably a cellulose synthase which is associated with the primary or the secondary plant cell wall of the species from which it has been derived.
Furthermore, the nucleic acid molecule according to this aspect of the invention may be derived from a monocotyledonous or dicotyledonous plant species. In a particularly preferred embodiment, the nucleic acid molecule is derived from Arabidopsis thaliana, Oryza sativa, wheat, barley, maize, Brassica ssp., Gossypium hirsutum (cotton) or Eucalyptus ssp., amongst others.
For the purposes of nomenclature, the nucleotide sequence shown in SEQ ID NO:1 relates to a cellulose gene as hereinbefore defined which comprises a cDNA sequence designated T20782 and which is derived from Arabidopsis thaliana. The amino acid sequence set forth in SEQ ID NO:2 relates to the polypeptide encoded by T20782.
The nucleotide sequence set forth in SEQ ID NO:3 relates to the nucleotide sequence of the complete Arabidopsis thaliana genomic gene RSW1, including both intron and exon sequences. The nucleotide sequence of SEQ ID NO:3 comprises exons 1-14 of the genomic gene and includes 2295 bp of 5xe2x80x2-untranslated sequences, of which approximately the first 1.9 kb comprises RSW1 promoter sequence (there is a putative TATA box motif at positions 1843-1850 of SEQ ID NO:3). The nucleotide sequence set forth in SEQ ID NO:3 is derived from the cosmid clone 23H12. This sequence is also the genomic gene equivalent of SEQ ID Nos:1 and 5.
The nucleotide sequence set forth in SEQ ID NO:4 relates to the partial nucleotide sequence of a genomic gene variant of RSW1, derived from cosmid clone 12C4. The nucleotide sequence of SEQ ID NO:4 comprises exon sequence 1-11 and part of exon 12 of the genomic gene sequence and includes 862 bp of 5xe2x80x2-untranslated sequences, of which approximately 700 nucleotides comprise RSW1 promoter sequences (there is a putative TATA box motif at positions 668-673 of SEQ ID NO:4). The genomic gene sequence set forth in SEQ ID NO:4 is the equivalent of the cDNA sequence set forth in SEQ ID NO:7 (i.e. cDNA clone Ath-A).
The nucleotide sequence shown in SEQ ID NO:5 relates to a cellulose gene as hereinbefore defined which comprises a cDNA equivalent of the Arabidopsis thaliana RSW1 gene set forth in SEQ ID NO:3. The amino acid sequence set forth in SEQ ID NO:6 relates to the polypeptide encoded by the wild-type RSW1 gene sequences set forth in SEQ ID Nos:3 and 5.
The nucleotide sequence shown in SEQ ID NO:7 relates to a cellulose gene as hereinbefore defined which comprises a cDNA equivalent of the Arabidopsis thaliana RSW1 gene set forth in SEQ ID NO:4. The nucleotide sequence is a variant of the nucleotide sequences set forth in SEQ ID Nos:3 and 5. The amino acid sequence set forth in SEQ ID NO:8 relates to the polypeptide encoded by the wild-type RSW1 gene sequences set forth in SEQ ID Nos:4 and 6.
The nucleotide sequence shown in SEQ ID NO:9 relates to a cellulose gene as hereinbefore defined which comprises a further wild-type variant of the Arabidopsis thaliana RSW1 gene set forth in SEQ ID Nos:3 and 5. The nucleotide sequence variant is designated Ath-B. The amino acid sequence set forth in SEQ ID NO:10 relates to the polypeptide encoded by the wild-type RSW1 gene sequence set forth in SEQ ID No:9.
The nucleotide sequence shown in SEQ ID NO:11 relates to a cellulose gene as hereinbefore defined which comprises a cDNA equivalent of the Arabidopsis thaliana rsw1 gene. The rsw1 gene is a mutant cellulose gene which produces a radial root swelling phenotype as described by Baskin et al (1992). The present inventors have shown herein that the rsw1 gene also produces reduced inflorescence length, reduced fertility, misshapen epidermal cells, reduced cellulose content and the accumulation of non-crystalline xcex2-1,4-glucan, amongst others, when expressed in plant cells. The rsw1 nucleotide sequence is a further variant of the nucleotide sequences set forth in SEQ ID Nos:3 and 5. The amino acid sequence set forth in SEQ ID NO:12 relates to the rsw1 polypeptide encoded by the mutant rsw1 gene sequence set forth in SEQ ID No:11.
The nucleotide sequence shown in SEQ ID NO:13 relates to a cellulose gene as hereinbefore defined which comprises a cDNA equivalent of the Oryza sativa RSW1 or RSW1-like gene. The nucleotide sequence is closely-related to the Arabidopsis thaliana RSW1 and rsw1 nucleotide sequences set forth herein (SEQ ID Nos:1, 3, 4, 5, 7, 9 and 11). The amino acid sequence set forth in SEQ ID NO:14 relates to the polypeptide encoded by the RSW1 or RSW1-like gene sequences set forth in SEQ ID No: 13.
Those skilled in the art will be aware of procedures for the isolation of further cellulose genes to those specifically described herein, for example further cDNA sequences and genomic gene equivalents, when provided with one or more of the nucleotide sequences set forth in SEQ ID Nos:1, 3, 4, 5, 7, 9, 11, or 13. In particular, hybridisations may be performed using one or more nucleic acid hybridisation probes comprising at least 10 contiguous nucleotides and preferably at least 50 contiguous nucleotides derived from the nucleotide sequences set forth herein, to isolate cDNA clones, mRNA molecules, genomic clones from a genomic library (in particular genomic clones containing the entire 5xe2x80x2 upstream region of the gene including the promoter sequence, and the entire coding region and 3xe2x80x2-untranslated sequences), and/or synthetic oligonucleotide molecules, amongst others. The present invention clearly extends to such related sequences.
The invention further extends to any homologues, analogues or derivatives of any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13.
A further aspect of the present invention contemplates a nucleic acid molecule which encodes or is complementary to a nucleic acid molecule which encodes, a polypeptide which is required for cellulose biosynthesis in a plant, such as cellulose synthase, and which is capable of hybridising under at least low stringency conditions to the nucleic acid molecule set forth in any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 1 1 or 13, or to a complementary strand thereof.
As an exemplification of this embodiment, the present inventors have shown that it is possible to isolate variants of the Arabidopsis thaliana RSW1 gene sequence set forth in SEQ ID NO:3, by hybridization under low stringency conditions. Such variants include related sequences derived from Gossypium hirsutum (cotton), Eucalyptus ssp. and A. thaliana. Additional variant are clearly encompassed by the present invention.
Preferably, the nucleic acid molecule further comprises a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, a cellulose synthase polypeptide, more preferably a cellulose synthase which is associated with the primary or secondary plant cell wall of the plant species from which said nucleic acid molecule was derived.
More preferably, the nucleic acid molecule according to this aspect of the invention encodes or is complementary to a nucleic acid molecule which encodes, a polypeptide which is required for cellulose biosynthesis in a plant, such as cellulose synthase, and which is capable of hybridising under at least medium stringency conditions to the nucleic acid molecule set forth in any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or to a complementary strand thereof.
Even more preferably, the nucleic acid molecule according to this aspect of the invention encodes or is complementary to a nucleic acid molecule which encodes, a polypeptide which is required for cellulose biosynthesis in a plant, such as cellulose synthase, and which is capable of hybridising under at least high stringency conditions to the nucleic acid molecule set forth in any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or to a complementary strand thereof.
For the purposes of defining the level of stringency, a low stringency is defined herein as being a hybridisation and/or a wash carried out in 6xc3x97SSC buffer, 0.1% (w/v) SDS at 28xc2x0 C. Generally, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash. A medium stringency comprises a hybridisation and/or a wash carried out in 0.2xc3x97SSC-2xc3x97SSC buffer, 0.1% (w/v) SDS at 42xc2x0 C. to 65xc2x0 C., while a high stringency comprises a hybridisation and/or a wash carried out in 0.1xc3x97SSC-0.2xc3x97SSC buffer, 0.1% (w/v) SDS at a temperature of at least 55xc2x0 C. Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of further clarification only, reference to the parameters affecting hybridisation between nucleic acid molecules is found in pages 2.10.8 to 2.10.16. of Ausubel et al. (1987), which is herein incorporated by reference.
In an even more preferred embodiment of the invention, the isolated nucleic acid molecule further comprises a sequence of nucleotides which is at least 40% identical to at least 10 contiguous nucleotides derived from any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a complementary strand thereof.
Still more preferably, the isolated nucleic acid molecule further comprises a sequence of nucleotides which is at least 40% identical to at least 50 contiguous nucleotides derived from the sequence set forth in any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a complementary strand thereof.
The present invention is particularly directed to a nucleic acid molecule which is capable of functioning as a cellulose gene as hereinbefore defined, for example a cellulose synthase gene such as, but not limited to, the Arabidopsis thaliana, Oryza sativa, wheat, barley, maize, Brassica ssp., Gossypium hirsutum or Eucalyptus ssp. cellulose synthase genes, amongst others. The subject invention clearly contemplates additional cellulose genes to those specifically described herein which are derived from these plant species.
The invention further contemplates other sources of cellulose genes such as but not limited to, tissues and cultured cells of plant origin. Preferred plant species according to this embodiment include hemp, jute, flax and woody plants including, but not limited to Pinus ssp., Populus ssp., Picea spp., amongst others.
A genetic sequence which encodes or is complementary to a sequence which encodes a polypeptide which is involved in cellulose biosynthesis may correspond to the naturally occurring sequence or may differ by one or more nucleotide substitutions, deletions and/or additions. Accordingly, the present invention extends to cellulose genes and any functional genes, mutants, derivatives, parts, fragments, homologues or analogues thereof or non-functional molecules but which are at least useful as, for example, genetic probes, or primer sequences in the enzymatic or chemical synthesis of said gene, or in the generation of immunologically interactive recombinant molecules.
In a particularly preferred embodiment, the cellulose genetic sequences are employed to identify and isolate similar genes from plant cells, tissues, or organ types of the same species, or from the cells, tissues, or organs of another plant species.
According to this embodiment, there is contemplated a method for identifying a related cellulose gene or related cellulose genetic sequence, for example a cellulose synthase or cellulose synthase-like gene, said method comprising contacting genomic DNA, or mRNA, or cDNA with a hybridisation effective amount of a first cellulose genetic sequence comprising any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a complementary sequence. homologue, analogue or derivative thereof derived from at least 10 contiguous nucleotides of said first sequence, and then detecting said hybridisation.
Preferably, the first genetic sequence comprises at least 50 contiguous nucleotides, even more preferably at least 100 contiguous nucleotides and even more preferably at least 500 contiguous nucleotides, derived from any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a complementary strand, homologue, analogue or derivative thereof.
The related cellulose gene or related cellulose genetic sequence may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell. Preferably, the related cellulose gene or related cellulose genetic sequence is derived from a plant species, such as a monocotyledonous plant or a dicotyledonous plant selected from the list comprising Arabidopsis thaliana, wheat, barley, maize, Brassica ssp., Gossypium hirsutum (cotton), Oryza sativa (rice), Eucalyptus ssp., hemp, jute, flax, and woody plants including, but not limited to Pinus ssp., Populus ssp., Picea spp., amongst others.
More preferably, related cellulose gene or related cellulose genetic sequence is derived from a plant which is useful in the fibre or timber industries, for example Gossypium hirsutum (cotton), hemp, jute, flax, Eucalyptus ssp. or Pinus ssp., amongst others. Alternatively, the related cellulose gene or related cellulose genetic sequence is derived from a plant which is useful in the cereal or starch industry, for example wheat, barley, rice or maize, amongst others.
In a particularly preferred embodiment, the first cellulose genetic sequence is labelled with a reporter molecule capable of giving an identifiable signal (e.g. a radioisotope such as 32P or 35S or a biotinylated molecule).
An alternative method contemplated in the present invention involves hybridising two nucleic acid xe2x80x9cprimer moleculesxe2x80x9d to a nucleic acid xe2x80x9ctemplate moleculexe2x80x9d which comprises a related cellulose gene or related cellulose genetic sequence or a functional part thereof, wherein the first of said primers comprises contiguous nucleotides derived from any one or more of SEQ ID Nos:1, 3, 4, 5. 7, 9, 11 or 13 or a homologue, analogue or derivative thereof and the second of said primers comprises contiguous nucleotides complementary to any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13. Specific nucleic acid molecule copies of the template molecule are amplified enzymatically in a polymerase chain reaction, a technique that is well known to one skilled in the art.
In a preferred embodiment, each nucleic acid primer molecule is at least 10 nucleotides in length, more preferably at least 20 nucleotides. in length, even more preferably at least 30 nucleotides in length, still more preferably at least 40 nucleotides in length and even still more preferably at least 50 nucleotides in length.
Furthermore, the nucleic acid primer molecules consists of a combination of any of the nucleotides adenine, cytidine, guanine, thymidine, or inosine, or functional analogues or derivatives thereof which are at least capable of being incorporated into a polynucleotide molecule without having an inhibitory effect on the hybridisation of said primer to the template molecule in the environment in which it is used.
Furthermore, one or both of the nucleic acid primer molecules may be contained in an aqueous mixture of other nucleic acid primer molecules, for example a mixture of degenerate primer sequences which vary from each other by one or more nucleotide substitutions or deletions. Alternatively, one or both of the nucleic acid primer molecules may be in a substantially pure form.
The nucleic acid template molecule may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell. Preferably, the nucleic acid template molecule is derived from a plant cell, tissue or organ, in particular a cell, tissue or organ derived from a plant selected from the list comprising Arabidopsis thaliana, Oryza sativa, wheat, barley, maize, Brassica ssp., Gossypium hirsutum and Eucalyptus ssp., hemp, jute, flax, and woody plants including, but not limited to Pinus ssp., Populus ssp., Picea spp., amongst others.
Those skilled in the art will be aware that there are many known variations of the basic polymerase chain reaction procedure, which may be employed to isolate a related cellulose gene or related cellulose genetic sequence when provided with the nucleotide sequences set forth in any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13. Such variations are discussed, for example, in McPherson et al (1991). The present invention extends to the use of all such variations in the isolation of related cellulose genes or related cellulose genetic sequences using the nucleotide sequences embodied by the present invention.
The isolated nucleic acid molecule according to any of the further embodiments may be cloned into a plasmid or bacteriophage molecule, for example to facilitate the preparation of primer molecules or hybridisation probes or for the production of recombinant gene products. Methods for the production of such recombinant plasmids, cosmids, bacteriophage molecules or other recombinant molecules are well-known to those of ordinary skill in the art and can be accomplished without undue experimentation. Accordingly, the invention further extends to any recombinant plasmid, bacteriophage, cosmid or other recombinant molecule comprising the nucleotide sequence set forth in any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a complementary sequence, homologue, analogue or derivative thereof.
The nucleic acid molecule of the present invention is also useful for developing genetic constructs which express a cellulose genetic sequence, thereby providing for the increased expression of genes involved in cellulose biosynthesis in plants, selected for example from the list comprising Arabidopsis thaliana, Oryza sativa, wheat, barley, maize, Brassica ssp., Gossypium hirsutum and Eucalyptus ssp., hemp, jute, flax, and woody plants including, but not limited to Pinus ssp., Populus ssp., Picea spp., amongst others. The present invention particularly contemplates the modification of cellulose biosynthesis in cotton, hemp, jute, flax, Eucalyptus ssp. and Pinus ssp. amongst others.
The present inventors have discovered that the genetic sequences disclosed herein are capable of being used to modify the level of non-crystalline xcex2-1,4,-glucan, in addition to altering cellulose levels when expressed, particularly when expressed in plants cells. In particular, the Arabidopsis thaliana rsw1 mutant has increased levels of non-crystalline xcex2-1,4,-glucan, when grown at 31xc2x0 C. compared to wild-type plants, grown under identical conditions. The expression of a genetic sequence described herein in the antisense orientation in transgenic plants grown at only 21xc2x0 C. is shown to reproduce many aspects of the rsw1 mutant phenotype. Accordingly, the present invention clearly extends to the modification of non-crystalline xcex2-1,4,-glucan biosynthesis in plants, selected for example from the list comprising Arabidopsis thaliana, Oryza sativa, wheat, barley, maize, Brassica ssp., Gossypium hirsutum and Eucalyptus ssp., hemp, jute, flax, and woody plants including, but not limited to Pinus ssp., Populus ssp., Picea spp., amongst others. The present invention particularly contemplates the modification of non-crystalline xcex2-1,4,-glucan biosynthesis in cotton, hemp, jute, flax, Eucalyptus ssp. and Pinus ssp., amongst others.
The present invention further extends to the production and use of non-crystalline xcex2-1,4-glucan and to the use of the glucan to modify the properties of plant cell walls or cotton fibres or wood fibres. Such modified properties are described herein (Example 13).
The inventors have discovered that the rsw1 mutant has altered carbon partitioning compared to wild-type plants, resulting in significantly higher starch levels therein. The isolated nucleic acid molecules provided herein are further useful for altering the carbon partitioning in a cell. In particular, the present invention contemplates increased starch production in transgenic plants expressing the nucleic acid molecule of the invention in the antisense orientation or alternatively, expressing a ribozyme or co-suppression molecule comprising the nucleic acid sequence of the invention.
The invention further contemplates reduced starch and/or non-crystalline xcex2-1,4-glucan product in transgenic plants expressing the nucleic acid molecule of the invention in the sense orientation such that cellulose production is increased therein.
Wherein it is desired to increase cellulose production in a plant cell, the coding region of a cellulose gene is placed operably behind a promoter, in the sense orientation, such that a cellulose gene product is capable of being expressed under the control of said promoter sequence. In a preferred embodiment, the cellulose genetic sequence is a cellulose synthase genomic sequence, cDNA molecule or protein-coding sequence.
In a particularly preferred embodiment, the cellulose genetic sequence comprises a sequence of nucleotides substantially the same as the sequence set forth in any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13 or a homologue, analogue or derivative thereof.
Wherein it is desirable to reduce the content of cellulose or to increase the content of non-crystalline xcex2-1,4-glucan, the nucleic acid molecule of the present invention is expressed in the antisense orientation under the control of a suitable promoter. Additionally, the nucleic acid molecule of the invention is also useful for developing ribozyme molecules, or in co-suppression of a cellulose gene. The expression of an antisense, ribozyme or co-suppression molecule comprising a cellulose gene, in a cell such as a plant cell, fungal cell, insect cell, animal cell, yeast cell or bacterial cell, may also increase the solubility, digestibility or extractability of metabolites from plant tissues or alternatively, or increase the availability of carbon as a precursor for any secondary metabolite other than cellulose (e.g. starch or sucrose). By targeting the endogenous cellulose gene, expression is diminished, reduced or otherwise lowered to a level that results in reduced deposition of cellulose in the primary or secondary cell walls of the plant cell, fungal cell, insect cell, animal cell, yeast cell or bacterial cell, and more particularly, a plant cell. Additionally, or alternatively, the content of non-crystalline xcex2-1,4-glucan is increased in such cells.
Co-suppression is the reduction in expression of an endogenous gene that occurs when one or more copies of said gene, or one or more copies of a substantially similar gene are introduced into the cell. The present invention also extends to the use of co-suppression to inhibit the expression of a gene which encodes a cellulose gene product, such as but not limited to cellulose synthase. Preferably, the co-suppression molecule of the present invention targets a plant mRNA molecule which encodes a cellulose synthase enzyme, for example a plant, fungus, or bacterial cellulose synthase mRNA, and more preferably a plant mRNA derived from Arabidopsis thaliana, Oryza saliva, wheat, barley, maize, Brassica ssp., Gossypium hirsutum and Eucalyptus ssp., hemp, jute, flax, or a woody plant such as Pinus ssp., Populus ssp., or Picea spp., amongst others.
In a particularly preferred embodiment, the gene which is targeted by a co-suppression molecule, comprises a sequence of nucleotides set forth in any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a complement, homologue, analogue or derivative thereof.
In the context of the present invention, an antisense molecule is an RNA molecule which is transcribed from the complementary strand of a nuclear gene to that which is normally transcribed to produce a xe2x80x9csensexe2x80x9d mRNA molecule capable of being translated into a polypeptide component of the cellulose biosynthetic pathway. The antisense molecule is therefore complementary to the mRNA transcribed from a sense cellulose gene or a part thereof. Although not limiting the mode of action of the antisense molecules of the present invention to any specific mechanism, the antisense RNA molecule possesses the capacity to form a double-stranded mRNA by base pairing with the sense mRNA, which may prevent translation of the sense mRNA and subsequent synthesis of a polypeptide gene product.
Preferably, the antisense molecule of the present invention targets a plant mRNA molecule which encodes a cellulose gene product, for example cellulose synthase. Preferably, the antisense molecule of the present invention targets a plant mRNA molecule which encodes a cellulose synthase enzyme, for example a plant mRNA derived from Arabidopsis thaliana, Oryza saliva, wheat, barley, maize, Brassica ssp., Gossypium hirsutum and Eucalyptus ssp., hemp, jute, flax, or a woody plant such as Pinus ssp., Populus ssp., or Picea spp., amongst others.
In a particularly preferred embodiment, the antisense molecule of the invention targets an mRNA molecule encoded by any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a homologue, analogue or derivative thereof.
Ribozymes are synthetic RNA molecules which comprise a hybridising region complementary to two regions, each of at least 5 contiguous nucleotide bases in the target sense mRNA. In addition, ribozymes possess highly specific endoribonuclease activity, which autocatalytically cleaves the target sense mRNA. A complete description of the function of ribozymes is presented by Haseloff and Gerlach (1988) and contained in International Patent Application No. WO89/05852.
The present invention extends to ribozyme which target a sense mRNA encoding a cellulose gene product, thereby hybridising to said sense mRNA and cleaving it, such that it is no longer capable of being translated to synthesise a functional polypeptide product. Preferably, the ribozyme molecule of the present invention targets a plant mRNA molecule which encodes a cellulose synthase enzyme, for example a plant mRNA derived from Arabidopsis thaliana, Gossypium hirsutum (cotton), Oryza sativa (rice), Eucalyptus ssp., hemp, jute, flax, or a woody plant such as Pinus ssp., Populus ssp., or Picea spp., amongst others.
In a particularly preferred embodiment, the ribozyme molecule will target an mRNA encoded by any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a homologue, analogue or derivative thereof.
According to this embodiment, the present invention provides a ribozyme or antisense molecule comprising at least 5 contiguous nucleotide bases derived from any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a complementary nucleotide sequence or a homologue, analogue or derivative thereof, wherein said antisense or ribozyme molecule is able to form a hydrogen-bonded complex with a sense mRNA encoding a cellulose gene product to reduce translation thereof.
In a preferred embodiment, the antisense or ribozyme molecule comprises at least 10 to 20 contiguous nucleotides derived from any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a complementary nucleotide sequence or a homologue, analogue or derivative thereof. Although the preferred antisense and/or ribozyme molecules hybridise to at least about 10 to 20 nucleotides of the target molecule, the present invention extends to molecules capable of hybridising to at least about 50-100 nucleotide bases in length, or a molecule capable of hybridising to a full-length or substantially full-length mRNA encoded by a cellulose gene, such as a cellulose synthase gene.
Those skilled in the art will be aware of the necessary conditions, if any, for selecting or preparing the antisense or ribozyme molecules of the invention.
It is understood in the art that certain modifications, including nucleotide substitutions amongst others, may be made to the antisense and/or ribozyme molecules of the present invention, without destroying the efficacy of said molecules in inhibiting the expression of a gene encoding a cellulose gene product such as cellulose synthase. It is therefore within the scope of the present invention to include any nucleotide sequence variants, homologues, analogues, or fragments of the said gene encoding same, the only requirement being that said nucleotide sequence variant, when transcribed, produces an antisense and/or ribozyme molecule which is capable of hybridising to a sense mRNA molecule which encodes a cellulose gene product.
Gene targeting is the replacement of an endogenous gene sequence within a cell by a related DNA sequence to which it hybridises, thereby altering the form and/or function of the endogenous gene and the subsequent phenotype of the cell. According to this embodiment, at least a part of the DNA sequence defined by any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a related cellulose genetic sequence, may be introduced into target cells containing an endogenous cellulose gene, thereby replacing said endogenous cellulose gene.
According to this embodiment, the polypeptide product of said cellulose genetic sequence possesses different catalytic activity and/or expression characteristics, producing in turn modified cellulose deposition in the target cell. In a particularly preferred embodiment of the invention, the endogenous cellulose gene of a plant is replaced with a gene which is merely capable of producing non-crystalline xcex2-1,4-glucan polymers or alternatively which is capable of producing a modified cellulose having properties similar to synthetic fibres such as rayon, in which the xcex2-1,4-glucan polymers are arranged in an antiparallel configuration relative to one another.
The present invention extends to genetic constructs designed to facilitate expression of a cellulose genetic sequence which is identical, or complementary to the sequence set forth in any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a functional derivative, part, homologue, or analogue thereof, or a genetic construct designed to facilitate expression of a sense molecule, an antisense molecule, ribozyme molecule, co-suppression molecule, or gene targeting molecule containing said genetic sequence.
The said genetic construct of the present invention comprises the foregoing sense, antisense, or ribozyme, or co-suppression nucleic acid molecule, or gene-targeting molecule, placed operably under the control of a promoter sequence capable of regulating the expression of the said nucleic acid molecule in a prokaryotic or eukaryotic cell, preferably a plant cell. The said genetic construct optionally comprises, in addition to a promoter and sense, or antisense, or ribozyme, or co-suppression, or gene-targeting nucleic acid molecule, a terminator sequence.
The term xe2x80x9cterminatorxe2x80x9d refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3xe2x80x2-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3xe2x80x2-end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants. Examples of terminators particularly suitable for use in the genetic constructs of the present invention include the nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) 35S gene, and the zein gene terminator from Zea mays. 
Reference herein to a xe2x80x9cpromoterxe2x80x9d is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. A promoter is usually, but not necessarily, positioned upstream or 5xe2x80x2, of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.
In the present context, the term xe2x80x9cpromoterxe2x80x9d is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of said sense, antisense, or ribozyme, or co-suppression nucleic acid molecule, in a plant cell. Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of a sense antisense, ribozyme or co-suppression molecule and/or to alter the spatial expression and/or temporal expression of said sense or antisense, or ribozyme, or co-suppression, or gene-targeting molecule. For example, regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence driving expression of a sense, or antisense, or ribozyme, or co-suppression, or gene-targeting molecule, thereby conferring copper inducibility on the expression of said molecule.
Placing a sense or ribozyme, or antisense, or co-suppression, or gene-targeting molecule under the regulatory control of a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5xe2x80x2 (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
Examples of promoters suitable for use in genetic constructs of the present invention include viral, fungal, bacterial, animal and plant derived promoters capable of functioning in prokaryotic or eukaryotic cells. Preferred promoters are those capable of regulating the expression of the subject cellulose genes of the invention in plants cells, fungal cells, insect cells, yeast cells, animal cells or bacterial cells, amongst others. Particularly preferred promoters are capable of regulating expression of the subject nucleic acid molecules in plant cells. The promoter may regulate the expression of the said molecule constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or plant pathogens, or metal ions, amongst others. Preferably, the promoter is capable of regulating expression of a sense, or ribozyme, or antisense, or co-suppression molecule or gene targeting, in a plant cell. Examples of preferred promoters include the CaMV 35S promoter, NOS promoter, octopine synthase (OCS) promoter and the like.
In a most preferred embodiment, the promoter is capable of expression in any plant cell, such as, but not limited to a plant selected from the list comprising Arabidopsis thaliana, Oryza saliva, wheat, barley, maize, Brassica ssp., Gossypium hirsutum and Eucalyptus ssp., hemp, jute, flax, and woody plants including, but not limited to Pinus ssp., Populus ssp., Picea spp., amongst others.
In a particularly preferred embodiment, the promoter may be derived from a genomic clone encoding a cellulose gene product, in particular the promoter contained in the sequence set forth in SEQ ID NO:3 or SEQ ID NO:4. Preferably, the promoter sequence comprises nucleotide 1 to about 1900 of SEQ ID NO:3 or nucleotides 1 to about 700 of SEQ ID NO:4 or a homologue, analogue or derivative capable of hybridizing thereto under at least low stringency conditions.
Optionally, the genetic construct of the present invention further comprises a terminator sequence.
In an exemplification of this embodiment, there is provided a binary genetic construct comprising the isolated nucleotide sequence of nucleotides set forth in SEQ ID NO:3. There is also provided a genetic construct comprising the isolated nucleotide sequence of nucleotides set forth in SEQ ID NO:1, in the antisense orientation, placed operably in connection with the CaMV 35S promoter.
In the present context, the term xe2x80x9cin operable connection withxe2x80x9d means that expression of the isolated nucleotide sequence is under the control of the promoter sequence with which it is connected, regardless of the relative physical distance of the sequences from each other or their relative orientation with respect to each other.
An alternative embodiment of the invention is directed to a genetic construct comprising a promoter or functional derivative, part, fragment, homologue, or analogue thereof, which is capable of directing the expression of a polypeptide early in the development of a plant cell at a stage when the cell wall is developing, such as during cell expansion or during cell division. In a particularly preferred embodiment, the promoter is contained in the sequence set forth in SEQ ID NO:3 or SEQ ID NO:4. Preferably, the promoter sequence comprises nucleotide 1 to about 1900 of SEQ ID NO:3 or nucleotides 1 to about 700 of SEQ ID NO:4 or a homologue, analogue or derivative capable of hybridizing thereto under at least low stringency conditions.
The polypeptide may be a reporter molecule which is encoded by a gene such as the bacterial xcex2-glucuronidase gene or chloramphenicol acetyltransferase gene or alternatively, the firefly luciferase gene. Alternatively, the polypeptide may be encoded by a gene which is capable of producing a modified cellulose in the plant cell when placed in combination with the normal complement of cellulose genes which are expressible therein, for example it may be a cellulose-like gene obtained from a bacterial or fungal source or a cellulose gene obtained from a plant source.
The genetic constructs of the present invention are particularly useful in the production of crop plants with altered cellulose content or structure. In particular, the rate of cellulose deposition may be reduced leading to a reduction in the total cellulose content of plants by transferring one or more of the antisense, ribozyme or co-suppression molecules described supra into a plant or alternatively, the same or similar end-result may be achieved by replacing an endogenous cellulose gene with an inactive or modified cellulose gene using gene-targeting approaches. The benefits to be derived from reducing cellulose content in plants are especially apparent in food and fodder crops such as, but not limited to maize, wheat, barley, rye, rice, barley, millet or sorghum, amongst others where improved digestibility of said crop is desired. The foregoing antisense, ribozyme or co-suppression molecules are also useful in producing plants with altered carbon partitioning such that increased carbon is available for growth, rather than deposited in the form of cellulose.
Alternatively, the introduction to plants of additional copies of a cellulose gene in the xe2x80x98sensexe2x80x99 orientation and under the control of a strong promoter is useful for the production of plants with increased cellulose content or more rapid rates of cellulose biosynthesis. Accordingly, such plants may exhibit a range of desired traits including, but not limited to modified strength and/or shape and/or properties of fibres, cell and plants, increased protection against chemical, physical or environmental stresses such as dehydration, heavy metals (e.g. cadmium) cold, heat or wind, increased resistance to attack by pathogens such as insects, nematodes and the like which physically penetrate the cell wall barrier during invasion/infection of the plant.
Alternatively, the production of plants with altered physical properties is made possible by the introduction thereto of altered cellulose gene(s). Such plants may produce xcex2-1,4-glucan which is either non-crystalline or shows altered crystallinity. Such plants may also exhibit a range of desired traits including but not limited to, altered dietary fibre content, altered digestibility and degradability or producing plants with altered extractability properties.
Furthermore, genetic constructs comprising a plant cellulose gene in the xe2x80x98sensexe2x80x99 orientation may be used to complement the existing range of cellulose genes present in a plant, thereby altering the composition or timing of deposition of cellulose deposited in the cell wall of said plant. In a preferred embodiment, the cellulose gene from one plant species or a xcex2-1,4-glucan synthase gene from a non-plant species is used to transform a plant of a different species, thereby introducing novel cellulose biosynthetic metabolism to the second-mentioned plant species.
In a related embodiment, a recombinant fusion polypeptide may be produced containing the active site from one cellulose gene product fused to another cellulose gene product, wherein said fusion polypeptide exhibits novel catalytic properties compared to either xe2x80x98parentxe2x80x99 polypeptide from which it is derived. Such fusion polypeptides may be produced by conventional recombinant DNA techniques known to those skilled in the art, either by introducing a recombinant DNA capable of expressing the entire fusion polypeptide into said plant or alternatively, by a gene-targeting approach in which recombination at the DNA level occurs in vivo and the resultant gene is capable of expressing a recombinant fusion polypeptide.
The present invention extends to all transgenic methods and products described supra, including genetic constructs.
The recombinant DNA molecule carrying the sense, antisense, ribozyme or co-suppression molecule of the present invention and/or genetic construct comprising the same, may be introduced into plant tissue, thereby producing a xe2x80x9ctransgenic plantxe2x80x9d, by various techniques known to those skilled in the art. The technique used for a given plant species or specific type of plant tissue depends on the known successful techniques. Means for introducing recombinant DNA into plant tissue include, but are not limited to, transformation (Paszkowski et al., 1984), electroporation (Fromm et al., 1985), or microinjection of the DNA (Crossway et al., 1986), or T-DNA-mediated transfer from Agrobacterium to the plant tissue. Representative T-DNA vector systems are described in the following references: An et al.(1985); Herrera-Estrella et al. (1983a,b); Herrera-Estrella et al. (1985). Once introduced into the plant tissue, the expression of the introduced gene may be assayed in a transient expression system, or it may be determined after selection for stable integration within the plant genome. Techniques are known for the in vitro culture of plant tissue, and in a number of cases, for regeneration into whole plants. Procedures for transferring the introduced gene from the originally transformed plant into commercially useful cultivars are known to those skilled in the art.
A still further aspect of the present invention extends to a transgenic plant such as a crop plant, carrying the foregoing sense, antisense, ribozyme, co-suppression, or gene-targeting molecule and/or genetic constructs comprising the same. Preferably, the transgenic plant is one or more of the following: Arabidopsis thaliana, Oryza sativa, wheat, barley, maize, Brassica ssp., Gossypium hirsutum and Eucalyptus ssp., hemp, jute, flax, Pinus ssp., Populus ssp., or Picea spp. Additional species are not excluded.
The present invention further extends to the progeny of said transgenic plant.
Yet another aspect of the present invention provides for the expression of the subject genetic sequence in a suitable host (e.g. a prokaryote or eukaryote) to produce full length or non-full length recombinant cellulose gene products.
Hereinafter the term xe2x80x9ccellulose gene productxe2x80x9d shall be taken to refer to a recombinant product of a cellulose gene as hereinbefore defined. Accordingly, the term xe2x80x9ccellulose gene productxe2x80x9d includes a polypeptide product of any gene involved in the cellulose biosynthetic pathway in plants, such as, but not limited to a cellulose synthase gene product.
Preferably, the recombinant cellulose gene product comprises an amino acid sequence having the catalytic activity of a cellulose synthase polypeptide or a functional mutant, derivative part, fragment, or analogue thereof.
In a particularly preferred embodiment of the invention, the recombinant cellulose gene product comprises a sequence or amino acids that is at least 40% identical to any one or more of SEQ ID Nos:2, 6, 8, 10, 12 or 14, or a homologue, analogue or derivative thereof.
Single and three-letter abbreviations used for amino acid residues contained in the specification are provided in Table 1.
In the present context, xe2x80x9chomologuesxe2x80x9d of an amino acid sequence refer to those polypeptides, enzymes or proteins which have a similar catalytic activity to the amino acid sequences described herein, notwithstanding any amino acid substitutions, additions or deletions thereto. A homologue may be isolated or derived from the same or another plant species as the species from which the polypeptides of the invention are derived.
xe2x80x9cAnaloguesxe2x80x9d encompass polypeptides of the invention notwithstanding the occurrence of any non-naturally occurring amino acid analogues therein.
xe2x80x9cDerivativesxe2x80x9d include modified peptides in which ligands are attached to one or more of the amino acid residues contained therein, such as carbohydrates, enzymes, proteins, polypeptides or reporter molecules such as radionuclides or fluorescent compounds. Glycosylated, fluorescent, acylated or alkylated forms of the subject peptides are particularly contemplated by the present invention. Additionally, derivatives of an amino acid sequence described herein which comprises fragments or parts of the subject amino acid sequences are within the scope of the invention, as are homopolymers or heteropolymers comprising two or more copies of the subject polypeptides. Procedures for derivatizing peptides are well-known in the art.
Substitutions encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as xe2x80x9cconservativexe2x80x9d, in which an amino acid residue contained in a cellulose gene product is replaced with another naturally-occurring amino acid of similar character, for example Gly←xe2x86x92Ala, Val←xe2x86x92Ile←xe2x86x92Leu, Asp←xe2x86x92Glu, Lys←xe2x86x92Arg, Asn←xe2x86x92Gln or Phe←xe2x86x92Trp←xe2x86x92Tyr.
Substitutions encompassed by the present invention may also be xe2x80x9cnon-conservativexe2x80x9d, in which an amino acid residue which is present in a cellulose gene product described herein is substituted with an amino acid with different properties, such as a naturally-occurring amino acid from a different group (eg. substituted a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.
Non-conventional amino acids encompassed by the invention include, but are not limited to those listed in Table 2.
Amino acid substitutions are typically of single residues, but may be of multiple residues, either clustered or dispersed.
Amino acid deletions will usually be of the order of about 1-10 amino acid residues, while insertions may be of any length. Deletions and insertions may be made to the N-terminus, the C-terminus or be internal deletions or insertions. Generally, insertions within the amino acid sequence will be smaller than amino- or carboxy-terminal fusions and of the order of 1-4 amino acid residues.
A homologue, analogue or derivative of a cellulose gene product as referred to herein may readily be made using peptide synthetic techniques well-known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Techniques for making substituent mutations at pre-determined sites using recombinant DNA technology, for example by M13 mutagenesis, are also well-known. The manipulation of nucleic acid molecules to produce variant peptides, polypeptides or proteins which manifest as substitutions, insertions or deletions are well-known in the art.
The cellulose gene products described herein may be derivatized further by the inclusion or attachment thereto of a protective group which prevents, inhibits or slows proteolytic or cellular degradative processes. Such derivatization may be useful where the half-life of the subject polypeptide is required to be extended, for ample to increase the amount of cellulose produced in a primary or secondary cell wall of a plant cell or alternatively, to increase the amount of protein produced in a bacterial or eukaryotic expression system. Examples of chemical groups suitable for this purpose include. but are not limited to, any of the non-conventional amino acid residues listed in Table 2, in particular a D-stereoisomer or a methylated form of a naturally-occurring amino acid listed in Table 1. Additional chemical groups which are useful for this purpose are selected from the list comprising aryl or heterocyclic N-acyl substituents, polyalkylene oxide moieties, desulphatohirudin muttons, alpha-muteins, alpha-aminophosphonic acids, water-soluble polymer groups such as polyethylene glycol attached to sugar residues using hydrazone or oxime groups, benzodiazepine dione derivatives, glycosyl groups such as beta-glycosylamine or a derivative thereof, isocyanate conjugated to a polyol functional group or polyoxyethylene polyol capped with diisocyanate, amongst others. Similarly, a cellulose gene product or a homologue, analogue or derivative thereof may be cross-linked or fused to itself or to a protease inhibitor peptide, to reduce susceptibility of said molecule to proteolysis.
In an alternative embodiment of the invention, the recombinant cellulose gene product is characterised by at least one functional xcex2-glycosyl transferase domain contained therein.
The term xe2x80x9cxcex2-glycosyl transferase domainxe2x80x9d as used herein refers to a sequence of amino acids which is highly conserved in different processive enzymes belonging to the class of glycosyl transferase enzymes (Saxena et al., 1995), for example the bacterial xcex2-1,4-glycosyl transferase enzymes and plant cellulose synthase enzymes amongst others, wherein said domain possesses a putative function in contributing to or maintaining the overall catalytic activity, substrate specificity or substrate binding of an enzyme in said enzyme class. The xcex2-glycosyl transferase domain is recognisable by the occurrence of certain amino acid residues at particular locations in a polypeptide sequence, however there is no stretch of contiguous amino acid residues comprised therein.
As a consequence of the lack of contiguity in a xcex2-glycosyl transferase domain, it is not a straightforward matter to isolate a cellulose gene by taking advantage of the presence of a xcex2-glycosyl transferase domain in the polypeptide encoded by said gene. For example, the xcex2-glycosyl transferase domain would not be easily utilisable as a probe to facilitate the rapid isolation of all xcex2-glycosyl transferase genetic sequences from a particular organism and then to isolate from those genetic sequences a cellulose gene such as cellulose synthase.
In a preferred embodiment, the present invention provides an isolated polypeptide which:
(i)contains at least one structural xcex2-glycosyl transferase domain as hereinbefore defined; and
(ii) has at least 40% amino acid sequence similarity to at least 20 contiguous amino acid residues set forth in any one or more of SEQ ID Nos:2, 6, 8, 10, 12 or 14, or a homologue, analogue or derivative thereof.
More preferably, the polypeptide of the invention is at least 40% identical to at least 50 contiguous amino acid residues, even more preferably at least 100 amino acid residues of any one or more of SEQ ID Nos:2, 6, 8, 10, 12 or 14, or a homologue, analogue or derivative thereof.
In a particularly preferred embodiment, the percentage similarity to any one or more of SEQ ID Nos:2, 6, 8, 10, 12 or 14 is at least 50-60%. more preferably at least 65-70%, even more preferably at least 75-80% and even more preferably at least 85-90%, including about 91% or 95%.
In a related embodiment, the present invention provides a xe2x80x9csequencably purexe2x80x9d form of the amino acid sequence described herein. xe2x80x9cSequencably purexe2x80x9d is hereinbefore described as substantially homogeneous to facilitate amino acid determination.
In a further related embodiment, the present invention provides a xe2x80x9csubstantially homogeneousxe2x80x9d form of the subject amino acid sequence, wherein the term xe2x80x9csubstantially homogeneousxe2x80x9d is hereinbefore defined as being in a form suitable for interaction with an immunologically interactive molecule. Preferably, the polypeptide is at least 20% homogeneous, more preferably at least 50% homogeneous, still more preferably at least 75% homogeneous and yet still more preferably at least about 95-100% homogenous, in terms of activity per microgram of total protein in the protein preparation.
The present invention further extends to a synthetic peptide of at least 5 amino acid residues in length derived from or comprising a part of the amino acid sequence set forth in any one or more of SEQ ID Nos:2, 6, 8, 10, 12 or 14, or having at least 40% similarity thereto.
Those skilled in the art will be aware that such synthetic peptides may be useful in the production of immunologically interactive molecules for the preparation of antibodies or as the peptide component of an immunoassay.
The invention further extends to an antibody molecule such as a polyclonal or monoclonal antibody or an immunologically interactive part or fragment thereof which is capable of binding to a cellulose gene product according to any of the foregoing embodiments.
The term xe2x80x9cantibodyxe2x80x9d as used herein, is intended to include fragments thereof which are also specifically reactive with a polypeptide of the invention. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as for whole antibodies. For example, F(abxe2x80x2)2 fragments can be generated by treating antibody with pepsin. The resulting F(abxe2x80x2)2 fragment can be treated to reduce disulfide bridges to produce Fabxe2x80x2 fragments.
Those skilled in the art will be aware of how to produce antibody molecules when provided with the cellulose gene product of the present invention. For example, by using a polypeptide of the present invention polyclonal antisera or monoclonal antibodies can be made using standard methods. A mammal, (e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the polypeptide which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a polypeptide include conjugation to carriers or other techniques well known in the art. For example, the polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired IgG molecules corresponding to the polyclonal antibodies may be isolated from the sera.
To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art. For example, the hybridoma technique originally developed by Kohler and Milstein (1975) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., 1983), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985), and screening of combinatorial antibody libraries (Huse el al., 1989). Hybridoma cells can be screened immunochemically for production of antibodies which are specifically reactive with the polypeptide and monoclonal antibodies isolated.
As with all immunogenic compositions for eliciting antibodies, the immunogenically effective amounts of the polypeptides of the invention must be determined empirically. Factors to be considered include the immunogenicity of the native polypeptide, whether or not the polypeptide will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier and route of administration for the composition, i.e. intravenous, intramuscular, subcutaneous, etc., and the number of immunizing doses to be administered. Such factors are known in the vaccine art and it is well within the skill of immunologists to make such determinations without undue experimentation.
It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody.
The present invention is further described by reference to the following non-limiting Figures and Examples.